This invention relates to nucleic acid and amino acid sequences of a human phospholipase A2 protein and to the use of these sequences in the diagnosis, prevention, and treatment of cancer and inflammation.
The secretory phospholipase A2 (PtA2) superfamily comprises a number of heterogeneous enzymes whose common feature is to hydrolyze the sn-2 fatty acid acyl ester bond of phosphoglycerides. Hydrolysis of the glycerophospholipids releases free fatty acids and lysophospholipids. PLA2 activity generates precursors for the biosynthesis of biologically active lipids, hydroxy fatty acids, and platelet-activating factor.
PLA2 hydrolysis of the sn-2 ester bond in phospholipids generates free fatty acids, such as arachidonic acid and lysophospholipids. Arachidonic acid is processed into bioactive lipid mediators of inflammation such as lyso-platelet-activating factor and eicosanoids. The synthesis of arachidonic acid from membrane phospholipids is the rate-limiting step in the biosynthesis of the four major classes of eicosanoids (prostaglandins, prostacyclins, thromboxanes and leukotrienes) involved in pain, fever, and inflammation. Furthermore, leukotriene-B4 is known to function in a feedback loop which further increases PLA2 activity (Wijkander, J. et al. (1995) J. Biol. Chem. 270:26543-26549).
PLA2s, first described as components of snake venoms and pancreatic juice and later defined in mammals, have traditionally been classified into several major groups and subgroups based on their amino acid sequence, disulfide bond location, and divalent cation requirements. The PLA2s of groups I, II, and m consist of low molecular weight, secreted, Ca2+-dependent proteins, Group IV PLA2s are primarily 85-kDa, Ca2+-dependent cytosolic phospholipases, and an additional group comprises the Ca2+-independent cytosolic phospholipases (Davidson, F. F. and Dennis, E. A., (1990) J. Mol. Evol. 31:228-238; and Dennis, E. F. (1994) J. Biol Chem. 269:13057-13060).
The first PLA2s to be extensively characterized were the Group I, II, and III PLA2s found in snake and bee venoms. These venom PLA2s share many features with mammalian PLA2s including a common catalytic mechanism, the same Ca+ requirement, and conserved primary and tertiary structures. In addition to their role in the digestion of prey, the venom PLA2s display neurotoxic, myotoxic, anticoagulant, and proinflammatory effects in mammalian tissues. This diversity of pathophysiological effects is due to the presence of specific, high affinity receptors for these enzymes on various cells and tissues (Lambeau, G. et al. (1995) J. Biol. Chem. 270:5534-5540).
At least four different groups of PLA2s have been characterized in mammalian cells, including Group I (pancreatic), Groups IIA, and IIC (inflammatory), and Group V (expressed in the heart). The pancreatic PLA2s function in the digestion of dietary lipids and have been proposed to play a role in cell proliferation, smooth muscle contraction, and acute lung injury. The inflammatory PLA2s are potent mediators of inflammatory processes and are highly expressed in serum and synovial fluids of patients with inflammatory disorders. These Group II PLA2s are found in most human cell types assayed and are expressed in diverse pathological processes such as septic shock, intestinal cancers, rheumatoid arthritis, and epidermal hyperplasia. A Group V PLA2 has been cloned from brain tissue and is strongly expressed in heart tissue. Other PLA2s have been cloned from various human tissues and cell lines, suggesting a large diversity of PLA2s. A human PLA2 was recently cloned from fetal lung, and based on its structural properties, appears to be the first member of a new group of mammalian PLAs, referred to as Group X. (Chen J. et al. (1994) J. Biol. Chem. 269:2365-2368; Kennedy, B. P., et al. (1995) J. Biol. Chem. 270:22378-22385; Komada, M., et al. (1990) Biochem. Biophys. Res. Commun. 168:1059-1065; and Cupillard, L. et al. (1997) J. Biol. Chem. 272:15745-15752).
The discovery of a new human phospholipase A2 protein and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cancer and inflammation.
The invention features a substantially purified polypeptide, human phospholipase A2 protein (PHPLA2), having the amino acid sequence shown in SEQ ID NO:1, or fragments thereof.
The invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or fragments thereof and a composition comprising said polynucleotide sequence. The invention also provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence SEQ ID NO:1, or fragments of said polynucleotide sequence. The invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, or fragments or variants of said polynucleotide sequence.
The invention also provides an isolated and purified sequence comprising SEQ ID NO:2 or variants thereof. In addition, the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:2. The invention also provides a polynucleotide sequence comprising the complement of SEQ ID NO:2, or fragments or variants thereof.
The present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences. In yet another aspect, the expression vector containing the polynucleotide sequence is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof, the method comprising the steps of:a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding PHPLA2 under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified PHPLA2 having the amino acid sequence of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention also provides a purified antagonist of the polypeptide of SEQ ID NO:1. In one aspect the invention provides a purified antibody which binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:1.
Still further, the invention provides a purified agonist of the polypeptide of SEQ ID NO:1.
The invention also provides a method for treating or preventing a cancer comprising administering to a subject in need of such treatment an effective amount of an antagonist to PHPLA2.
The invention also provides a method for treating or preventing inflammation comprising administering to a subject in need of such treatment an effective amount of an antagonist to PHPLA2.
The invention also provides a method for detecting a polynucleotide which encodes PHPLA2 in a biological sample comprising the steps of:a) hybridizing the complement of the polynucleotide sequence which encodes SEQ ID NO:1 to nucleic acid of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding PHPLA2 in the biological sample. In one aspect the nucleic acid of the biological sample is amplified by the. polymerase chain reaction prior to hybridization.